Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkL(f)) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkL(f) and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased omega-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 +/- 7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 +/- 1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.